The long term goal of this study is to determine the molecular mechanisms by which endogenous mouse mammary tumor virus (MuMTV) is involved in mammary tumorigenesis in the C3Hf mouse. We have detected the presence of new MuMTV-specific endogenous proviral DNA integrated into the cellular DNA of 12/12 C3Hf mammary tumors studied. Moreover, in 7 of these 12 tumors both Hind III and Eco RI digests have generated similar size host-viral junction fragments suggesting that integration of this new endogenous MuMTV proviral DNA occurs at common sites. Integration of proviral DNA at identical locations in different tumors suggests that the promotor insertion model proposed for avian leukemogenesis may be applicable for mammary tumorigenesis in C3Hf mice. The aim of this proposal is to determine if the promotor insertion model is occurring in C3Hf mammary tumorigenesis. To determine if proviral DNA integration sites are the same in different C3Hf tumors we will characterize the cellular sequences present in the similar size host-viral junction fragments by detailed restriction enzyme mapping and by hybridization to probes of cloned cellular sequences found in the various viral-host junction fragments. The viral-host junction fragments will be cloned using recombinant DNA technology and the celullar sequences present subcloned so that we will have available sufficient and specific DNA for detailed restriction enzyme mapping and for the preparation of hybridization probes. The promotor insertion model predicts that tumors should contain new mRNAs initiated by a viral promotor (located within the newly integrated proviral LTR) and containing the 5 foot terminal region of the viral DNA and adjacent cellular sequences. We will be examining C3Hf tumors for the presence of novel mRNAs which hybridize to an MuMTV-specific cDNA5 foot probe (but not to a cDNArep probe) and which may also hybridize to cellular sequences cloned from the host-viral junction fragments. In addition, in order to understand what role these integration sites may play in C3Hf tumor induction we will determine if the cellular sequences at these integration sites are hypomethylated, DNase I sensitive, or transcribed into mRNAs in normal organs before proviral DNA integration.